TLR2 antagonistic antibody and use thereof

ABSTRACT

The present invention is directed to a cross-reactive antibody, which specifically inhibits or blocks the mammalian Toll-like receptor 2 (TLR2)-mediated immune cell activation. The invention is further directed to an isolated nucleic acid or vector coding for the variable regions of the heavy and/or light chain of the antibody. It is further providing a pharmaceutical composition comprising the antibody, nucleic acid or vector and is directed to the use of the composition in the prevention and/or treatment of inflammatory processes or any other process induced by bacterial infection, trauma, or chronic inflammation, or for the prevention and/or treatment of bacteriaemia or sepsis.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/595,204, filed Jan. 25, 2008, now U.S. Pat. No. 8,623,353, whichapplication is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/EP2004/010700, filed Sep. 23, 2004, whichwas published under PCT Article 21(2) in English, the entire contents ofwhich are incorporated herein by reference.

The present invention is directed to a cross-reactive antibody, whichspecifically inhibits or blocks the mammalian Toll-like receptor 2(TLR2)-mediated immune cell activation. The invention is furtherdirected to an isolated nucleic acid or vector coding for the variableregions of the heavy and/or light chain of said antibody. It is furtherproviding a pharmaceutical composition comprising said antibody, nucleicacid or vector and is directed to the use of said composition in theprevention and or treatment of inflammatory processes or any otherprocess induced by bacterial infection, trauma, or chronic inflammation,or for the prevention and/or treatment of bacteriaemia or sepsis.

Host cells recognize specific patterns of microbial components throughToll-like receptors (TLRs) which are crucial in mediating innate immuneresponses^(1,2). Lipopolysaccharide (LPS) from the outer membrane ofGram-negative bacteria is a potent agonist for TLR4 whose effects on thehost organism have been studied extensively in experimental models ofinfection and septic shock³⁻⁵. Over-stimulation of host immune cells bymicrobial products accompanied by the release of large amounts ofinflammatory mediators is recognized as a major cause of septicshock⁶⁻⁹.

Indeed, this concept has been validated by using both gene targeted micelacking the expression of the respective receptors, and by receptorspecific inhibition of microbial product induced host cell activation.For example, the non-redundant role of CD14 as an important element of acellular LPS recognition system has been demonstrated by application ofinhibitory anti CD14 antibodies in rabbits^(10,11). The blockade of LPSreceptors or extracellular effector proteins as the earliest possibletargets of therapeutic strategies was shown to be preventive¹². Anotherapproach of therapeutic intervention in septic shock has beeninterference with the functions of proinflammatory cytokines such asTNFα or IL-1β. For instance, competitive inhibition of cytokine bindingto its signaling receptors by application of recombinant extracellulardomain (ECD) or naturalizing receptor antagonist proteins have beenshown to be protective in LPS induced shock in rats¹³. In addition,antagonistic antibodies targeting cytokines or ECDs of its receptorshave been tested¹⁴. While cytokine blockade for therapeutic interventionin acute infections (sepsis) has been disappointing¹⁵, its use intreatment of chronic inflammations is promising^(16,17).

Besides Gram-negative, Gram-positive bacteria lacking LPS play animportant role in the clinical manifestation of shock⁸. Cell wallcomponents from these bacteria such as peptidoglycan (PGN) andlipoteichoic acid (LTA) are considered the main causative agents ofGram-positive shock^(18,19). PGN is a main component of Gram-positive,but also of other bacterial cell walls, and consists of an alternating β(1,4) linked N-acetylmuramyl and N-acetylglucosaminyl glycan crosslinked by small peptides²⁰. In contrast, the macroamphiphile LTA, asaccharide chain molecule consisting of repetitive oligosaccharidesconnected by alcohols such as ribitol and carrying acyl chains throughwhich it is anchored to the bacterial cytoplasmic membrane, is specificfor Gram-positive bacteria²¹. For example, LTA has been described tocarry the major stimulatory activity of Bacillus subtilis ²². Thestimulatory properties of tripalmityolated proteins whose production isnot restricted to Gram-negative or Gram-positive bacteria are mimickedby the synthetic compoundN-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)-3-lysine(P₃CSK₄)²³.

Most of these bacterial products are known to activate innate immunecell responses by triggering the TLR2 signaling cascade². The TLR2ECD,whose N-terminal portion has been implicated in PGN recognition²⁴,contains an array of distinct leucine rich repeat (LRR) motifs²⁵. TheLRR rich domain is followed by an LRR C-terminal (LRRCT), atrans-membrane, and an intracellular C-terminal Toll-IL-1 receptor-plantdisease resistance protein (TIR) domain²⁵.

WO 01/36488 generally describes an anti-TLR2 antibody. It is furtherdescribed to prepare such an antibody by using a TLR2 immunogen, forexample a part of the TLR2 molecule, for example a domain (e.g. theextracellular domain) or a shorter peptide or polypeptide sequence e.g.an epitopic sequence. However, it is indicated in WO 01/36488 that it ispreferred for the immunogen to be one which mimics the natural proteinas far as possible.

WO 01/36488 in particular obtains and discloses a monoclonal antibodyhaving particular properties and characteristics (e.g. binding andinhibition characteristics). This antibody is called, “Mab TL2.1”. Thehybridoma producing this antibody was deposited under the terms of theBudapest Treaty at the European Collection of Cell Cultures (ECACC) onOct. 28, 1999 under the Accession No. 99102832. This antibody is theonly precise and fully disclosed Example for an antibody presented in WO01/36488.

The inventors conducted some research work and tested Mab TL2.1 for itscharacteristics and it turned out that this antibody is notcross-reactive between different mammalian species.

Therefore, it is the objective problem underlying the present inventionto provide a cross-reactive antibody, which specifically inhibits orblocks the mammalian Toll-like receptor 2 (TLR2)-mediated immune cellactivation by specifically binding to the highly conserved C-terminalportion of the extracellular domains of TLR2. It is further an objectiveproblem of the present invention to provide an antibody which is capableof prevention and/or treatment of bacteriaemia or sepsis, or ofprevention and/or treatment of inflammatory processes or any otherprocess induced by bacterial infection, trauma, or chronic inflammation.Additionally, a problem underlying the invention is to provide animproved approach of gene therapy, based on an overexpression of theantibody or a functional part thereof in a host organism in order toachieve the above mentioned therapeutic effect.

These objects are solved by the subject-matters of the independentclaims. Preferred embodiments are set forth in the dependent claims.

Herein, it is shown that a murine monoclonal antibody (mAb) raisedagainst the murine TLR2 extracellular domain (=ECD) inhibits TLR2mediated activation of murine and human cells. The protective potentialof neutralizing TLR2 with this antibody was demonstrated in vivo usingtwo TLR2 dependent shock models. Thus, antibody targeting of the TLR2ECDis a valuable strategy to prevent TLR2 driven septic shock.

In this invention, for the first time a cross-reactive antibody isdescribed, which is capable of inhibiting or blocking the mammalianToll-like receptor 2 (TLR2)-mediated immune cell activation byspecifically binding to the C-terminal portion of the extracellulardomains of at least human and murine TLR2. The cross-reactivity of theantibody of the invention in particular was shown by specificity for thehuman and murine TLR2 sequences. Thus, the antibody of the presentinvention for the first time is capable of specifically binding to thehighly conserved TLR2 sequence shared among most mammals, in particularamong mice and human beings.

It is noted that according to experimental data obtained by theinventors, the presently known antibodies as disclosed in WO 01/36488(TL2.1) interact with human TLR2 and an unspecific antigen from murinecells in FACS analysis and immunocytochemical staining analysis whereasthe antibody of the present invention (e.g. T2.5) specificallyrecognizes both murine and human TLR2, therefore being cross-reactive.The respective experimental data are provided in the Figures and theExamples.

The present invention in particular is directed to the following aspectsand embodiments:

According to a first aspect, the present invention is directed to across-reactive antibody, which specifically inhibits or blocks themammalian Toll-like receptor 2 (TLR2)-mediated immune cell activation byspecifically binding to the C-terminal portion of the extracellulardomains of at least human and murine TLR2.

As mentioned above, the antibody of the present invention is defined asbeing cross-reactive as an essential feature. The term “cross-reactive”as used herein refers to the ability of an individual antibody to reactwith an antigenic determinant within the TLR2 orthologue products of twodifferent species (here: human and murine). Cross reactivity in themeaning of the present invention arises because one specific antigen hasan epitope which is structurally similar to the analogous epitope withinthe analogous protein of two species. As mentioned above, an example ofcross-reactivity in the meaning of the present invention is thecapability of an anti-TLR2 antibody to specifically bind to theC-terminal portion of the extracellular domains of at least human andmurine TLR2 which is preferably encompassing a region from amino acidsT221 to R587 (for the corresponding sequence information it is referredto Genebank accession number HSU 88878) or a subregion thereof.

The term extracellular domain is used herein as commonly defined in thecorresponding field of science, i.e. as usually defined in the field oftype I transmembrane receptors. Those are proteins that span thethickness of the plasma membrane of the cell, with one end of thereceptor outside (extracellular domain) and one inside (intracellulardomain) the cell. Both are connected by the transmembrane domain.

The antibody of the present invention is, according to an embodiment, apolyclonal antibody, a monoclonal antibody, a humanized antibody, achimeric antibody, or a synthetic antibody.

The term “antibody”, is used herein for intact antibodies as well asantibody fragments, which have a certain ability to selectively bind toan epitop. Such fragments include, without limitations and as anexample, Fab, F(ab′)₂ und Fv antibody fragment. The term “epitop” meansany antigen determinant of an antigen, to which the paratop of anantibody can bind. Epitop determinants usually consist of chemicallyactive surface groups of molecules (e.g. amino acid or sugar residues)and usually display a three-dimensional structure as well as specificphysical properties.

The antibodies according to the invention can be produced according toany known procedure. For example, the pure complete TLR2ECD or a part ofit can be produced and used as immunogen, to immunize an animal and toproduce specific antibodies.

The production of polyclonal antibodies is commonly known. Detailedprotocols can be found for example in Green et al, Production ofPolyclonal Antisera, in Immunochemical Protocols (Manson, editor), pages1-5 (Humana Press 1992) und Coligan et al, Production of PolyclonalAntisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols InImmunology, section 2.4.1 (1992). In addition, the expert is familiarwith several techniques regarding the purification and concentration ofpolyclonal antibodies, as well as of monoclonal antibodies (Coligan etal, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).

The production of monoclonal antibodies is as well commonly known.Examples include the hybridoma method (Kohler and Milstein, 1975,Nature, 256:495-497, Coligan et al., section 2.5.1-2.6.7; and Harlow etal., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub.1988)), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole, et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96).

In brief, monoclonal antibodies can be attained by injecting a mixturewhich contains the protein in question into mice. The antibodyproduction in the mice is checked via a serum probe. In the case of asufficient antibody titer, the mouse is sacrificed and the spleen isremoved to isolate B-cells. The B cells are fused with myeloma cellsresulting in hybridomas. The hybridomas are cloned and the clones areanalyzed. Positive clones which contain a monoclonal antibody againstthe protein are selected and the antibodies are isolated from thehybridoma cultures. There are many well established techniques toisolate and purify monoclonal antibodies. Such techniques includeaffinity chromatography with protein A sepharose, size-exclusionchromatography and ion exchange chromatography. Also see for example,Coligan et al, section 2.7.1-2.7.12 and section, “Immunglobulin G(IgG)”, in Methods In Molecular Biology, volume 10, pages 79-104 (HumanaPress 1992).

According to a preferred embodiment, the antibody of the inventionspecifically binds through its variable regions of the heavy- and lightchain, which regions carry the amino acid sequence as depicted in SEQ IDNO:1 and/or 2, or a variant thereof. The term “variant” as used in thisconnection is explained in detail below (regarding the aspect directedto the nucleic acid of the invention).

Methods of making such antibody fragments, and synthetic and derivatisedantibodies are well known in the art. Also included in the scope of theterm, “antibody” as used herein (in addition to the ones indicatedabove) are antibody fragments containing the complementarity-determiningregions (CDRs) or hypervariable regions of the antibodies. These may bedefined as the region comprising the amino acid sequences on the lightand heavy chains of an antibody which form the three-dimensional loopstructure that contributes to the formation of the antigen binding site.CDRs may be used to generate CDR-grafted antibodies. The term “CDRgrafted” defines an antibody having an amino acid sequence in which atleast parts of one or more sequences in the light and/or variabledomains have been replaced by analogous parts of CDR sequences from anantibody having a different binding specificity for a given antigen.

One of skill in the art can readily produce such CDR grafted antibodiesusing methods well known in the art (see Borrebaeck, AntibodyEngineering: A Practical Guide, W. H. Freeman and Company, New York,1992).

A chimeric antibody of the present invention may be prepared bycombining the variable region of an anti TLR2 antibody (or a partthereof) of one species according to the present invention with theconstant regions of an antibody derived from a different species. Achimeric antibody may be constructed, for example, according to methodsdescribed by Shaw et al., in J. Immun. 1987, 138: 4534 and Sun et al.,in PNAS USA, 1987, 84: 214-218.

Monoclonal antibodies and their fragments and derivatives are preferredantibodies according to the present invention.

A further aspect of the invention is thus a hybridoma or cell-lineproducing an antibody of the invention as defined above.

According to a further embodiment, the antibody of the invention islinked to a pharmaceutical agent and/or to a detectable agent.

In a second aspect, the present invention is directed to an isolatednucleic acid coding for the variable regions of the heavy and/or lightchain of the antibody as defined above.

In particular, the invention is directed to an isolated nucleic acidwhich comprises the sequence of SEQ ID NO: 1 and/or 2 or variantsthereof, wherein the variants are each defined as having one or moresubstitutions, insertions and/or deletions as compared to the sequenceof SEQ ID NO: 1 and/or 2, provided that said variants hybridize undermoderately stringent conditions to a nucleic acid which comprises thesequence of SEQ ID NO: 1 and/or 2, and further provided that saidvariants code for an amino acid having activity as a variable region ofan antibody specifically binding to the C-terminal portion of theextracellular domains of at least human and murine TLR2 or provided thatsaid variants comprise nucleic acid changes due to the degeneracy of thegenetic code, which code for the same or a functionally equivalent aminoacid as the nucleic acid sequence of SEQ ID NO: 1 and/or 2.

More preferably, the isolated nucleic acid of the invention comprises atleast the sequence of nucleic acids No. 172-201, 244-294 and/or 385-417of SEQ ID NO: 1, or of nucleic acids No. 130-174, 220-240 and/or 337-363of SEQ ID NO: 2, or parts thereof. The above mentioned sequencescomprise complementarity determining regions (CDR's) 1, 2 and 3,respectively.

Regarding SEQ ID NO: 1, more precisely, nucleic acids No. 172-195,247-270 and 385-417 or parts thereof are preferred (CDR 1-3), which wereidentified by application of the IMGT database. Alternatively, nucleicacids 187-201 and 244-294 (CDR 1 and 2) are preferred, identified by theV-BASE database.

Regarding SEQ ID NO: 2, more precisely, nucleic acids No. 139-168,220-228 and 337-363 or parts thereof are preferred (CDR 1-3), which wereidentified by application of the IMGT database. Alternatively, nucleicacids 130-174 and 220-240 (CDR 1 and 2) are preferred, identified by theV-BASE database.

A chart comprising SEQ ID NO: 1 and its complementary sequence as wellas the encoded amino acids (SEQ ID NO: 6) are depicted below showing thenucleic acid and amino acid sequence of the heavy chain's variableregion of the antibody of the invention.

SEQ ID NO: 1 1 to 501        10         20         30         40         50ATGTCCTCTC CACAGTCCCT GAAGACACTG ATTCTAACCA TGGGATGGAGTACAGGAGAG GTGTCAGGGA CTTCTGTGAC TAAGATTGGT ACCCTACCTCMetSerSer ProGlnSerLeu LysThrLeu IleLeuThr MetGlyTrpSer>        60         70         80         90        100CTGGATCTTT CTCTTCCTCC TGTCAGGAAC TGCAGGTGTC CACTCCCAGGGACCTAGAAA GAGAAGGAGG ACAGTCCTTG ACGTCCACAG GTGAGGGTCCTrpIlePhe  LeuPheLeu LeuSerGlyThr AlaGlyVal HisSerGln>       110        120        130        140        150TTCAGCTGCA GCAGTCTGGA CCTGAGCTGG TGAACCCTGG GGCGTCAGTGAAGTCGACGT CGTCAGACCT GGACTCGACC ACTTGGGACC CCGCAGTCACValGlnLeuGln GlnSerGly ProGluLeu ValAsnProGly AlaSerVal>       160        170        180         190       200AAGTTGTCCT GCAAGGCTTC T

 

TTCAACAGGA CGTTCCGAAG ACCGAAGTGG AAGTGTTGGA TGCCATATTTLysLeuSer CysLysAlaSer 

 

       210        220        230        240        250CTGGGTGAAG CAGGGGCCTG GACAGGGACT TGAGTGGATT GGA

GACCCACTTC GTCCCCGGAC CTGTCCCTGA ACTCACCTAA CCTACCTAAA TrpValLys GlnGlyPro GlyGlnGlyLeu GluTrpIle Gly

        260       270        280        290        300

AAGGCC TAGGATCTCT ACCATCATGA TTGAAGTTAC TCTTAAAGTT CCTGTTCCGG

LysAla>        310        320        330        340        350GCATTGACTG TAGACACATC CTCCAGCACA GCGTACATGG AACTCCACAGCGTAACTGAC ATCTGTGTAG GAGGTCGTGT CGCATGTACC TTGAGGTGTCAlaLeuThr ValAspThrSer SerSerThr AlaTyrMet GluLeuHisSer>       360        370        380        390        400CCTGACATCT GAAGACTCTG CGGTCTATTT CTGT

 

GGACTGTAGA CTTCTGAGAC GCCAGATAAA GACACGTTTCT GACTGACCACLeuThrSer GluAspSer AlaValTyrPhe Cys

 

        410        420       430        440        450

 

TGG GGCCAGGGCA CCACTCTCAC AGTCTCCTCACCTGTAAGGA ACTGATAACC CCGGTCCCGT GGTGAGAGTG TCAGAGGAGT

 

Trp GlyGlnGly ThrThrLeuThr ValSerSer>       460        470        480        490        500GCCAAAACGA CACCCCCATC TGTCTATCCA CTGGCCCCTG GATCTGCTGCCGGTTTTGCT GTGGGGGTAG ACAGATAGGT GACCGGGGAC CTAGACGACGAlaLysThr ThrProProSer ValTyrPro LeuAlaPro GlySerAlaAla> C G

It is noted that the underlined regions are CDR1 and CDR2 sequentiallydefined by usage of V-BASE database, both nucleic acid and amino acidsequences are underlined accordingly. Italic/bold regions are CDR 1 to 3defined by application of IMGT database, both nucleic acid and aminoacid sequences are marked accordingly (applies also to SEQ ID NO: 2).

Further, a chart comprising SEQ ID NO: 2 and its complementary sequenceas well as the encoded amino acids (SEQ ID NO: 7) are depicted belowshowing the nucleic acid and amino acid sequence of the light chain'svariable region of the antibody of the invention.

SEQ ID NO: 2 1 to 443        10         20         30         40         50ATGGAGTCAG ACACACTCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGGTACCTCAGTC TGTGTGAGGA CGATACCCAC GACGACGAGA CCCAAGGTCCMetGluSer AspThrLeuLeu LeuTrpVal LeuLeuThr TrpValProGly>        60         70         80         90        100CTCCACTGGT GACATTGTGC TCACCCAATC TCCAGCTTCT TTGGCTGTGTGACCTGACCA CTGTAACACG AGTGGGTTAG AGGTCGAAGA AACCGACACASerThrGly  AspIleVal LeuThrGlnSer ProAlaSer LeuAlaVal>       110        120        130        140        150CTCTAGGGCA GAGAGCCACC ATCTCCTGC

  GAGATCCCGT CTCTCGGTGG TAGAGGACGT CTCGGTCACT TTCACAACTTSerLeuGlyGln ArgAlaThr IleSerCys 

       160        170        180        190        200

TGGTAC CAACAGAAAC CAGGACAGCCATAATACCGT GTTCAAATTA CGTCACCATG GTTGTCTTTG GTCCTGTCGG

TrpTyr GlnGlnLys ProGlyGlnPro>       210        220        230        240        250ACCCAAACTC CTCATCTTT

 GGGGTCCCTG TGGGTTTGAG GAGTAGAAAC CACGTAGGTT GCATCTTAGA CCCCAGGGACProLysLeu LeuIlePhe 

 GlyValPro>        260        270        280        290        300TCAGGTTCAG TGGCAGTGGG TCTGGGACAG ACTTCAGCCT CAACATCCATAGTCCAAGTC ACCGTCACCC AGACCCTGTC TGAAGTCGGA GTTGTAGGTAValArgPheSer GlySerGly SerGlyThr AspPheSerLeu AsnIleHis>       310        320        330        340        350CCTGTGGAGG AGGATGATAT TGTAATGTAT TTCTGTC

 

GGACACCTCC TCCTACTATA ACATTACATA AAGACAGTCG TTTCATCCTTProValGlu GluAspAspIle ValMetTyr PheCys

 

       360        370        380        390        400

 

TCGGTG GAGGCACCAA GCTGGAAATC AAACGGGCTGTGAAGGCACC TGCAAGCCAC CTCCGTGGTT CGACCTTTAG TTTGCCCGAC

 

PheGly GlyGlyThrLys LeuGluIle LysArgAla>       410        420        430        440ATGCTGCACC AACTGTATCC ATCTTCCCAC CATCCAGTGA GCATACGACGTGG TTGACATAGG TAGAAGGGTG GTAGGTCACT CGTAspAlaAlaPro ThrValSer IlePhePro ProSerSerGlu Xxx>

Furthermore, the whole sequence of the variable region of the heavychain (including a 5′ non coding region) reads as follows:

SEQ ID NO: 3: CATGGACTGAAGGAGTAGAAAGACAACCTATGGCCAATGTCCTCTCCACAGTCCCTGAAGACACTGATTCTAACCATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAACCCTGGGGCGTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTTCACCTTCACAACCTACGGTATAAACTGGGTGAAGCAGGGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTAGTACTAACTTCAATGAGAATTTCAAGGACAAGGCCGCATTGACTGTAGACACATCCTCCAGCACAGCGTACATGGAACTCCACAGCCTGACATCTGAAGACTCTGCGGTCTATTTCTGTGCAAGACTGACTGGTGGGACATTCCTTGACTATTGGGGCCAGGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCC

The whole sequence of the variable region of the light chain (includinga 5′ non coding region) reads as follows:

SEQ ID NO: 4: CATGGACTGAAGGAGTAGAAAATCCTCTCATCTAGCTCTCAGAGATGGAGTCAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGCTCCACTGGTGACATTGTGCTCACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGTGAAAGTGTTGAATATTATGGCACAAGTTTAATGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTTTGGTGCATCCAACGTAGAATCTGGGGTCCCTGTCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGGAGGATGATATTGTAATGTATTTCTGTCAGCAAAGTAGGAAACTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGACCA

The nucleic acid variants according to the invention also comprisenucleic acid fragments which contain more than 10, preferably more than15, more than 20, more than 25 or more than 30 and up to 50 nucleotides.Most preferably, the fragments comprise the CDR regions as indicatedabove.

According to the state of the art an expert can test which derivativesand possible variations derived from these revealed nucleic acidsequences according to the invention are, are partially or are notappropriate for specific applications.

Amplification and detection methods are according to the state of theart. The methods are described in detail in protocol books which areknown to the expert. Such books are for example Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, andall subsequent editions. PCR-methods are described for example inNewton, PCR, BIOS Scientific Publishers Limited, 1994 and all subsequenteditions.

As defined above, “variants” are according to the invention especiallysuch nucleic acids, which contain one or more substitutions, insertionsand or deletions when compared to the nucleic acids of SEQ ID NO: 1 and2. These lack preferably one, but also 2, 3, 4, or more nucleotides 5′or 3′ or within the nucleic acid sequence, or these nucleotides arereplaced by others.

The nucleic acid sequences of the present invention comprise also suchnucleic acids which contain sequences in essence equivalent to thenucleic acids described in SEQ ID NO: 1 and 2. According to theinvention nucleic acids can show for example at least about 80%, moretypically at least about 90% or 95% sequence identity to the nucleicacids described in SEQ ID NO: 1 and 2.

It is noted that the above considerations also apply to SEQ ID NO: 3 and4.

The term “nucleic acid sequence” means a heteropolymer of nucleotides orthe sequence of these nucleotides. The term “nucleic acid”, as hereinused, comprises RNA as well as DNA including cDNA, genomic DNA andsynthetic (e.g. chemically synthesized) DNA and other polymer linkedbases such as PNA (peptide nucleic acids).

The invention comprises—as mentioned above—also such variants whichhybridize to the nucleic acids according to the invention undermoderately stringent conditions.

Stringent hybridization and wash conditions are in general the reactionconditions for the formation of duplexes between oligonucleotides andthe desired target molecules (perfect hybrids) or that only the desiredtarget can be detected. Stringent washing conditions mean, e.g., 0.2×SSC(0.03 M NaCl, 0.003 M sodium citrate, pH 7)/10.1% SDS at 65° C. Forshorter fragments, e.g. oligonucleotides up to 30 nucleotides, thehybridization temperature is below 65° C., for example at 50° C.,preferably above 55° C., but below 65° C. Stringent hybridizationtemperatures are dependent on the size or length, respectively of thenucleic acid and their nucleic acid composition and will beexperimentally determined by the skilled artisan. Moderately stringenthybridization temperatures are for example 42° C. and washing conditionswith 0.2×SSC/0.1% SDS at 42° C.

Also contemplated herein are amino acid sequences of the aboveidentified kind, which also encompass all sequences differing from theherein disclosed sequences by amino acid insertions, deletions, andsubstitutions.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a polypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity. This does notrequire more than routine experiments for the skilled artisan.

According to a further embodiment, the isolated nucleic acid of theinvention further comprises a nucleic acid specifying one or moreregulatory sequences operably linked thereto.

In a further embodiment, the present invention includes a vectorcomprising a nucleic acid. This vector is preferably an expressionvector which contains a nucleic acid according to the invention and oneor more regulatory nucleic acid sequences.

Numerous vectors are known to be appropriate for the transformation ofbacterial cells, for example plasmids and bacteriophages, like the phageX, are frequently used as vectors for bacterial hosts. Viral vectors canbe used in mammalian and insect cells to express exogenous DNAfragments, e.g. SV 40 and polyoma virus.

The transformation of the host cell can be done alternatively directlyusing “naked DNA” without the use of a vector.

The antibody/antibody fragment according to the invention can beproduced either in eukaryotic or prokaryotic cells. Examples foreukaryotic cells include mammalian, plant, insect and yeast cells.Appropriate prokaryotic cells include Escherichia coli and Bacillussubtilis.

Preferred mammalian host cells are CHO, COS, HeLa, 293T, HEH or BHKcells or adult or embryonic stem cells.

Alternatively, the antibody/antibody fragment according to the inventioncan be produced in transgenic plants (e.g. potatoes, tobacco) or intransgenic animals, for example in transgenic goats or sheep.

According to a preferred embodiment, the vector is a plasmid or viralsuch as an engineered retrovirus or adeno virus.

According to a third aspect, the invention provides a pharmaceuticalcomposition comprising an active component, preferably an antibody, anucleic acid or a vector as defined hereinabove.

The active components of the present invention are preferably used insuch a pharmaceutical composition in doses mixed with a pharmaceuticallyacceptable carrier or carrier material, that the disease can be treatedor at least alleviated. Such a composition can (in addition to theactive component and the carrier) include filling material, salts,buffer, stabilizers, solubilizers and other materials, which are knownstate of the art.

The term “pharmaceutically acceptable” is defined as non-toxic material,which does not interfere with effectiveness of the biological activityof the active component. The choice of the carrier is dependent on theapplication.

The pharmaceutical composition can contain additional components whichenhance the activity of the active component or which supplement thetreatment. Such additional components and/or factors can be part of thepharmaceutical composition to achieve a synergistic or additionaleffects or to minimize adverse or unwanted effects.

Thus, the pharmaceutical composition of the invention may furthercontain one or more pharmaceutically active ingredients in order tosupplement the treatment.

Preferably, such pharmaceutically active ingredients are selected fromantibiotic agents, antiinflammatory agents, and/or agents blockingfurther pattern recognition receptors. Those agents preferably arespecific for CD14, LBP, MD-2, TLR3, TLR4, TLR5, TLR7, TLR8, and/or TLR9.

Techniques for the formulation or preparation and application/medicationof compositions of the present invention are published in “Remington'sPharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latestedition. A therapeutically effective dose relates to the amount of acompound which is sufficient to improve the symptoms, for example atreatment, healing, prevention or improvement of such conditions. Anappropriate application can include for example oral, dermal, rectal,transmucosal or intestinal application and parenteral application,including intramuscular, subcutaneous, intramedular injections as wellas intrathecal, direct intraventricular, intravenous, intraperitoneal orintranasal injections. The intravenous injection is the preferredtreatment of a patient.

A typical composition for an intravenous infusion can be produced suchthat it contains 250 ml sterile Ringer solution and for example 1000 mgof the antibody of the invention. See also Remington's PharmaceuticalScience (15. edition, Mack Publishing Company, Easton, Ps., 1980).

The active component or mixture of it in the present case can be usedfor prophylactic and/or therapeutic treatments.

An amount which is adequate to reach the aforesaid effect is defined as“therapeutically effective dose”. Amounts, which are effective for theseapplications, depend on the severity of the condition and the generalcondition of the patient. However, the dose range is usually between 1and 100 mg antibody per kilogram body weight of the patient in need ofthe treatment. Systemic application of a specific antibody of theinvention, i.e. T2.5 (see experimental part) upon lipopeptide challengeinhibited inflammatory mediator release such as TNFalpha and preventedlethal shock-like syndrome in mice. 20 mg/kg of T2.5 was sufficient toprotect mice and administration of 40 mg/kg of T2.5 was protective even3 h after start of otherwise lethal challenge with Bacillus subtilis.Therefore, for treating mammals a dose of between 10 to 60 mg and morepreferably between 20 to 40 mg per kilogram body weight is appropriate.Single or multiple applications after a daily, weekly or monthlytreatment regimen can be performed with application rate and sampleschosen by the physician in charge. These applications largely dependfrom the question, whether a chronic or acute condition is to beprevented or treated. An individual dose is administered as a singledose to the mammal suffering from an acute infection, wherein theindividual dose is administered repeatedly to a mammal suffering from achronic infection and/or inflammation.

The invention is additionally directed to a hybridoma which produces amonoclonal antibody as defined herein.

The antibody, nucleic acid, vector or composition of the presentinvention preferably are used in the prevention and/or treatment ofinflammatory processes or any other process induced by bacterialinfection, trauma, or chronic inflammation. Furthermore, they can beused for the prevention and/or treatment of bacteriaemia or sepsis. Thechronic infection preferably is selected from rheumatoid or vasculararthritis or inflammatory bowel disease.

The present invention in particular is directed to a gene therapyapproach for use in the treatment of chronic diseases. The approachbasically follows the already known protocols for gene therapy andcomprises in particular the step of cloning a sequence comprising thevariable domains of the antibody of the invention as specified aboveinto an expression vector and introducing said expression vector into ahost, for example a human patient in order to cause an overexpression ofsaid antibody/antibody fragment in said patient.

According to a fourth aspect, the present invention is further directedto a screening method for identifying an antagonist capable ofinhibiting or blocking TLR2, comprising the steps of:

-   -   (a) generating or providing mammalian TLR2,    -   (b) contacting said TLR2 with a candidate compound,    -   (c) detecting the inhibition or blocking of said compound by a        suitable detection method,    -   (d) selecting a compound that has been tested positive in step        (c),    -   (e) optionally repeating steps (a)-(d) with a suitably modified        form of the compound of step (d).

The present invention is in additionally directed to the followingembodiments:

-   1. An antagonist, which specifically inhibits or blocks the    mammalian, preferably human, Toll-like receptor 2 (TLR2).-   2. The antagonist of emb. 1, which is an antibody, small molecule or    an aptamer.-   3. The antibody of emb. 2, which is a polyclonal antibody, a    monoclonal antibody, a humanized antibody, a chimeric antibody, or a    synthetic antibody.-   4. The antibody of emb. 3, which is directed against the    extracellular domain of TLR2.-   5. The use of an antagonist of one or more of emb. 1-4 in the    prevention and/or treatment of acute and/or chronic inflammatory    processes induced by bacterial infection.-   6. A screening method for identifying an antagonist capable of    inhibiting or blocking TLR2, comprising the steps of:    -   (f) generating or providing mammalian TLR2,    -   (g) contacting said TLR2 with a candidate antagonist,    -   (h) detecting the inhibition or blocking of said candidate        antagonist by a suitable detection method,    -   (i) selecting a candidate antagonist that has been tested        positive in step (c),    -   (j) optionally repeating steps (a)-(d) with a suitably modified        form of the candidate antagonist of step (d).

The present invention will be further described with reference to thefollowing figures and examples; however, it is to be understood that thepresent invention is not limited to such figures and examples.

FIG. 1 Application of mAb T2.5 for specific detection of TLR2. Resultsof flow-cytometry of HEK293 cells stably overexpressing Flag-taggedmTLR2 (a) or human TLR2 (b), as well as primary TLR2^(−/−) (c) andwild-type murine macrophages (d) by staining with mAb T2.5 (bold line,unfilled area). Negative controls represent cells not stained with aprimary, but incubated with a mouse IgG specific secondary antibody(filled areas). For positive controls, Flag-(a and b) and mTLR2 (c andd) specific polyclonal antisera were used (normal line, unfilled area).For immunoprecipitation with T2.5, lysates of HEK293 cellsoverexpressing murine or human TLR2, as well as of murine RAW264.7macrophages were applied as indicated (e). TLR2 precipitates werevisualized by application of Flag (HEK293) or mTLR2 (RAW264.7) specificpolyclonal antisera. Flag specific (αFlag) and protein G beads in theabsence of antibodies (pG), as well as vector transfected HEK293 cellswere used as controls. The size of TLR2 was 97 kDa.

FIG. 2 Subcellular localization of TLR2 in vitro. MAb T2.5 was used forcytochemical detection of overexpressed murine and human TLR2 (a), aswell as endogenous murine (TLR2^(+/+), wild-type) or human TLR2 inprimary macrophages (b). Vector transfected HEK293 cells, as well asTLR2^(−/−) primary macrophages were analyzed as staining controls.Concanavalin A was used for staining of cytoplasmic membranes. The barsin the lower right corners of each field represent a distance of 20 μm(a) or 10 μm (b) on the slides analyzed.

FIG. 3 Inhibitory effect of mAb T2.5 on cell activation in vitro. NF-κBdependent luciferase activities in HEK293 cells overexpressing eithermurine (a) or human TLR2 (b), as well as TNFα concentrations insupernatants of RAW264.7 (c) or primary murine macrophages (d)challenged with inflammatory stimulants are shown (ND, not detectable).Cells were incubated either with T2.5 or conT2 only (empty bars), oradditionally challenged with IL-1β (a, b, horizontally hatched bars),ultra pure LPS (c, d, bold upward hatched bars), P₃CSK₄ (filled bars),or h. i. B. subtilis (downward hatched bars, a to d). MAb and challenge(P₃CSK₄, LPS) dependent NF-κB/p65 nuclear translocation in humanmacrophages (e) was analyzed by cytochemical staining (Unstim.,unstimulated). NF-κB dependent electro mobility shift assay (EMSA) andphosphorylation of MAP kinases Erk1/2 (pErk1/2) and p38 (pP38), as wellas Akt (pAkt) were analyzed by applying nuclear or total extracts,respectively, from RAW624.7 macrophages (f, g). Cells were preincubatedwith the amounts of mAb T2.5 or conT2 indicated (μg/ml) and subsequentlychallenged with P₃CSK₄ or LPS for 90 min (f, arrows indicate specificNF-κB-DNA complexes) or 30 min (g, P38, specific immunoblot as positivecontrol). Untreated cells (control) were analyzed as controls.

FIG. 4 TLR2 expression in vivo. Flow-cytometry of splenocytes andperitoneal washout cells from wild-type and TLR2^(−/−) mice ex vivoimmediately upon isolation (n=5, cells pooled for each sample). CD11b⁺splenocytes from mice challenged with LPS for 24 h were analyzed forsurface and intracellular TLR2 expression (a) by staining with T2.5(bold line, TLR2^(+/+); filled area, TLR2^(−/−)). For analysis of TLR2regulation upon infection (b, c), mice were either left uninfected (−)or infected with Gram-positive B. subtilis and sacrificed after 24 h(+). Upon staining of CD11b, cells were stained with T2.5 (TLR2) eitherwithout (b) or upon permeabilization (c). Numbers in quadrants representthe proportion of single or double stained cells, respectively, ascompared to the total number of viable cells analyzed (%).

FIG. 5 Inhibitory effect of mAb T2.5 on host activation by microbialchallenge in vivo. Mice were pretreated i. p. with 1 mg mAb T2.5 (solidbars) or left untreated (empty bars). Mice were challenged after 1 hwith P₃CSK₄ and D-galactosamine (i. p.), as well as sacrificed 2 h or 4h later (n=4 for each group at each time point). Serum concentrations ofTNFα (a), GROα/KC (human IL-8 homologue) (b), IL-6 (c), and IL-12p40 (d)were analyzed by ELISA (*p<0.05, **p<0.005, ***p<0.001, student's t-testfor unconnected samples).

FIG. 6 Effects of mAb T2.5 administration on viability uponTLR2-specific systemic challenging. IFNγ and D-galactosamine sensitizedmice received either no mAb, 1 mg of mAb T2.5, or 1 mg of conT.2 i. p.30 min prior to microbial challenge with bacterial lipopeptide analogueP₃CSK₄ (a, open circles, no mAb, n=4; open triangles, mAb conT2, n=3;filled squares, mAb T2.5, n=4). Mice challenged with a high dose of h.i. B. subtilis were left untreated or treated 1 h later with dosages ofmAb T2.5 indicated (b, filled diamonds, 1 mg, n=3; open squares, 0.5 mg,n=3; open triangles, 0.25 mg, n=4; x's, 0.13 mg, n=4; open circles, nomAb T2.5, n=4) or with 1 mg of Abs as indicated at different time points(c, d). Administration of TLR2-specific mAb prior (−), as well as after(+) bacterial challenge (c, filled inverted triangles, no mAb, n=8; opencircles, mAb conT2, −1 h, n=3; filled diamonds, mAb T2.5, −1 h, n=4;open squares, mAb T2.5, +1 h, n=3; x's, mAb T2.5, +2 h, n=3; opendiamonds, mAb T2.5, +3 h, n=4; open triangles, mAb T2.5, +4 h, n=3).Administration of TLR2-specific mAb T2.5 prior (−) to bacterialchallenge (d, n=3 for experimental groups: open triangles, no mAb;filled squares, mAb T2.5, −3 h; open diamonds, mAb T2.5, −4 h; opencircles, mAb T2.5, −5 h; filled inverted triangles, mAb T2.5, −6 h).

FIG. 7 depicts a FACS analysis demonstrating that T2.5 specificallyrecognizes both murine and human TLR2, while TL2.1 interacts with humanTLR2 and an unspecific antigen from murine cells. FACS was performedusing 5% NGS and 1% Fcblock as blocking agents, with 5 μg/ml primaryantibody (TLR2.1 and T2.5), and 3.5 μg/ml FITCGoat anti-mouse IgG (Fab)as secondary antibody.

FIGS. 8 and 9 depict immunocytochemistry experiments demonstrating thatT2.5 specifically recognizes both murine and human TLR2, while TL2.1interacts with human TLR2 and a nonspecific antigen from murine cells in1HC analysis. Immunocytochemistry was performed as follows:

-   1. Seed 1×10⁵ Cells/cover glass/ml/well on 24-well plate, culture    overnight.-   2. Fix cells with Methanol at −20 degree for 8 minutes.-   3. Wash with PBS for 3 times (dip into 3 beakers containing PBS).-   4. Block with 2% NGS (normal goat serum in PBS) at 37 degree for 20    minutes in a humid chamber (20-30 μl for each slide).-   5. Incubate with T2.5/TL2.1 (5 μg/ml) in 2% NGS at 37 degree for 60    minutes in a humid chamber (20-30 μl for each slide).-   6. Wash with PBS for 3 times (dip into 3 beakers containing PBS).-   7. Incubate with secondary antibody (AlexaFluoro546 conjugated goat    anti mouse IgG, cat, A-11030, 8 μg/ml, Molecular Probes, Leiden,    Netherlands) in 2% NGS at 37 degree for 60 minutes in a humid    chamber (20-30 μl for each slide).

Option: Incubate with AlexaFluoro488 conjugated Concanavalin A for cellsurface staining (AlexaFluoro488 conjugated Concanavalin A, cat.C-11252, 25 μg/ml, Molecular Probes, Leiden, Netherlands) in 2% NGS at37 degree for 60 minutes in a humid chamber together with secondaryantibody (20-30 μl for each slide).

-   8. Wash with PBS for 3 times (dip into 3 beakers containing PBS).-   9. Dry at RT for 20 minutes in dark, mount in mounting reagent,    observe with confocol microscope (LSM510, Carl Zeiss, Oberkochen,    Germany).

FIGS. 10 to 12 Dose dependent inhibition of cell activation by T2.5 butnot by TL2.1 in both THP1 and RAW cells.

In order to analyze potential TLR2-inhibitory difference of TL2.1 andT2.5, human THP1 and murine RAW264.7 cells were used. Doses of bothTL2.1 and T2.5 ranging from 5 μg/ml to 40 μg/ml as indicated in thefigure were applied 30 min prior to challenge with P₃CSK₄ at indicatedconcentrations. TNFα and IL-6 concentrations in supernatants of THP1 andRAW264.7 cells were analyzed 24 h after start of challenge.

FIG. 13 Dose dependent binding of T2.5 to mTLR2ECD versus failed bindingof TL2.1 to the same antigen in ELISA.

ELISA plates were coated with anti human IgG Fc antibody, human IgGFcfused mouse TLR2ECD protein was then immobilized after BSA blocking andwashing followed by detection with T2.5 and TL2.1 at variousconcentrations as indicated. Specific signals were visualized viaapplication of HRP conjugated anti mouse IgG after primary antibodyincubation followed by reaction with HRP substrates.

FIG. 14 TL2.1 lacks binding capacity to overexpressed murine and humanTLR2 in ELISA while T2.5 binds both in the same assay.

ELISA plates were coated with anti Flag antibody, Flag fused mouse andhuman TLR2 and control proteins were then immobilized after BSA blockingand washing followed by detection with T2.5 and TL2.1 at 1 μg/ml asindicated. Specific signals were visualized via application of HRPconjugated anti mouse IgG after primary antibody incubation followed byreaction with HRP substrates.

FIG. 15 Lack of interaction between TL2.1 and murine TLR2 inImmunoprecipitation and subsequent immunoblot analysis.

Lysates of 1×10⁶ murine RAW264.7 macrophages or HEK293 cells, 1 μg ofeach antibody as indicated, and 20 μl of protein G beads (Santa Cruz,Calif., USA) were mixed for o. n. precipitation. Immune complexes wereanalyzed by immunoblot analysis with either Flag- or murineTLR2-specific antiserum.

FIG. 16: Antibodies (Abs) for immunoprecipitation (IP), as indicated,hT2.5 stands for linearized T2.5 heavy chain and light chain fused withC terminal human IgG1 Fc. Antigens (Ags) for IP: for lanes 1, 3, 5, 7,9, lysates from HEK 293 cells overexpressing Flag-tagged mouse TLR2, forlanes 2, 4, 6, 8, 10, lysates from HEK293 cells without anytransfection. A Western Blot (WB) was done with anti-Flag polyclonalsera. Binds at 97 kD resolved on 10% SDS-PAGE gel indicate the positionof Flag tagged murine TLR2 upon overexpression in HEK 293 cellsprecipitated with Abs.

FIG. 16A1-3:

A single chain (FIG. 16A1) and partially humanized TLR2-specific (hT2.5,FIG. 16A3) antibody was generated by subcloning of a mammalianexpression construct in the vector pcDNA3.1 within which expression isdriven by the CMV promoter (FIG. 16A2). From the 5′ end of the MCSsequence encoding a portion of the variable heavy chain (base residue 1to 450, see SEQ ID NO: 1), was followed successively by a linkersequence encoding 15 amino acids (Gly and Ser), a sequence encoding aportion of the variable light chain (base residue 61 to 396, see SEQ IDNO: 2), as well as the cds of the human Fcg (gamma) chain forming the 3′terminus (C terminus of the expressed hT2.5 protein) of the construct.

FIG. 17:

Molecular analysis of mAb T2.5 effects on TLR2ECD-P₃CSK₄ interaction.Binding of recombinant TLR2ECD-Fc fusion protein (T2EC, positivecontrols) to immobilized P₃CSK₄ upon pre-incubation with T2.5(T2EC+T2.5) at different molar excesses (a, ×1, ×3.3, ×10) or with anisotype matched control mAb (T2EC+con) at 10 fold molar excess only (b,×10). Binding was continuously monitored in a SPR biosensor device andamounts of antibodies used to gain high molecular excess over T2EC(co-incubation) were applied alone as negative controls (a, T2.5; b,con). Response units (RU) at 300 s are a measure for P₃CSK₄-bindingcapacities of T2EC and T2EC+mAb. For analysis of approximatelocalization of T2.5 epitope within the TLR2ECD, a mutant human TLR2construct lacking the N-terminal third of the LRR-rich ECD domain(hTLR2-mutH) was used for NF-κB dependent luciferase assay upontransient transfection, preincubation with mAb (T2.5, conT2), and P₃CSK₄challenge (c, solid bars). Absence of mAb treatment (no mAb) and/orP₃CSK₄ challenge (empty bars), as well as empty vector (Vector)represent respective controls (c).

FIG. 18:

Inhibitory effect of recombinant single chain and partially humanizedhT2.5 antibody on lipopeptide induced cell activation. HT2.5 wasoverexpressed in HEK293 cells and supernatant collected (see. FIG. 16).HEK293 cells were either transfected with reporter plasmids only (openbars) or cotransfected with human TLR2 (black bars). Three groups ofsamples were established for both TLR2″ and TLR2⁺ cells as indicated. Tosamples of the first group, regular supernatant of normal HEK293 cellswas administered. In the second group, cells were pretreated withculture supernatants of HEK293 cells overexpressing hT2.5. As positivecontrol, application of 50 μg/ml of T2.5 to supernatants in group threewas performed. Specifically, cells of the groups one and two werepretreated by four times removal of supernatant and subsequent additionof conditioned supernatant for 10 min each time while group threesamples were treated by a single application of T2.5 to the originalsupernatants. One set of all samples was left untreated while a secondset of samples was treated by application of lipopeptide analogue(P3CSK4) at a concentration of 100 ng/ml 40 minutes after antibody (T2.5or hT2.5) incubation. NF-κB dependent reportergene activation wasanalyzed 6 h upon start of lipopeptide challenge.

Result: The inhibitory effect of recombinant hT2.5 was similar to thatof native T2.5 on TLR2-dependent cell activation.

Abbreviations used herein: TLR, Toll-like receptor; mAb, monoclonalantibody; P₃CSK₄, tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine; h. i. B.subtilis, heat inactivated Bacillus subtilis; sPGN, solublepeptidoglycan; LTA, lipoteichoic acid

Examples

Application of Murine mAb T2.5 for Expression Analysis In Vitro

The inventors selected an IgG1κ anti TLR2 mAb named T2.5 whichspecifically recognized TLR2. HEK293 cells stably expressing murine orhuman TLR2 were stained specifically on their surface by T2.5 (FIGS. 1aand b ). Furthermore, T2.5 did not bind to primary murine TLR2^(−/−),but bound to wild-type macrophages cultured in vitro (FIGS. 1c and d ).T2.5 immuno-precipitated native murine and human TLR2 from lysates ofHEK293 cells overexpressing each of the two TLR2 orthologs (FIG. 1e ).Most importantly, T2.5 precipitated endogenous TLR2 from lysates ofRAW264.7 macrophages (FIG. 1e ). Next, the inventors analyzed T2.5 forits capacity to specifically detect TLR2 on the subcellular level.Overexpressed murine and human TLR2 (FIG. 2a ), as well as endogenousTLR2 were detectable in primary murine and CD14⁺ leukocyte derived humanmacrophages (FIG. 2b ).

Inhibitory Effects of T2.5 on TLR2 Specific Cell Activation

T2.5 inhibited murine and human TLR2 mediated cell activation by TLR2specific stimuli P₃CSK₄ or B. subtilis applied to HEK293 cellsoverexpressing TLR2, murine RAW264.7, and primary macrophages asmeasured by NF-κB dependent reporter gene assay and IL-8 specific enzymelinked immuno sorbent assay (ELISA), as well as TNFα and IL-6 specificELISA, respectively (FIG. 3a to d and data not shown). A second newlygenerated IgG1κ anti TLR2 mAb, conT2, was used as a control. This mAbbinds native murine (m) TLR2 in a manner comparable to T2.5 but nothuman TLR2 (data not shown) and failed to inhibit TLR2 dependent cellactivation (FIG. 3). Also, no inhibition of 1′-1 receptor or TLR4signaling by T2.5 was detected, indicating that TLR2 independentsignaling pathways in T2.5 treated cells remain intact (FIG. 3a to d ).Moreover, TLR2 mediated nuclear translocation of NF-κB was specificallyinhibited by T2.5 in human macrophages (FIG. 3e ). NF-κB specificelectro mobility shift assay (EMSA), as well as anti phospho p38,Erk1/2, and Akt immunoblot analysis revealed T2.5 but not conT2 dosedependent inhibition of P₃CSK₄ induced NF-κB-DNA binding and cellularkinase phosphorylation (FIG. 3f and g ).

Flow-Cytometry of Intracellular and Surface TLR2 Expression Ex Vivo

Since LPS induces TLR2 expression in primary macrophages in vitro (datanot shown), the inventors first compared T2.5 specific staining ofCD11b⁺ splenocytes from LPS challenged wild-type and TLR2^(−/−) mice byflow-cytometry. Weak surface staining and pronounced intracellularstaining were seen (FIG. 4a ). In subsequent experiments, peritonealcells and splenocytes from mice infected with the Gram-positivebacterium B. subtilis were analyzed. While surface expression of TLR2 inprimary murine macrophages was relatively strong upon in vitro culture(FIG. 1d ), surface expression was weak or not detectable inunchallenged CD11b⁺, CD11c⁺, CD19⁺, and GR1⁺ subpopulations ofsplenocytes and peritoneal washout cells (FIG. 4a, b and data notshown). Upon microbial challenge, however, TLR2 surface expressionstrongly increased in CD11b⁺ and GR1⁺ cells (FIG. 4b and data notshown). Signals were specific as tested by analysis of TLR2^(−/−) cells(FIG. 4). Again, intracellular staining of TLR2 revealed significantlevels of intracellular TLR2 expression which increased to a higherdegree than surface expression upon microbial challenge (FIG. 4a and b).

Antibody Mediated Interference with TLR2 Specific Immune ResponsesTowards Systemic Challenge

Next, the inventors determined cytokine and chemokine serumconcentrations in mice, either pretreated, or not pretreated with T2.5,as well as challenged with P₃CSK₄. While cytokine and chemokineconcentrations were low in sera of untreated mice (see methods), serumlevels of TNFα, IL-8, IL-6, and IL-12p40 were significantly lower inmice preinjected with T2.5 as compared to controls and measured afterchallenge (FIG. 5a to d ).

Both, a high dose (microbial product only) and a low dose model(additional sensitization with D-galactosamine) have been establishedfor bacterial product induced shock in mice²⁶. In order to interfere ina strictly defined model of septic shock, the inventors applied thebacterial lipopeptide analogue and TLR2 agonist P₃CSK₄ uponsensitization of mice with interferon gamma (IFNγ) andD-galactosamine²⁷. Sensitization was used to mimic priming of a hostdefense towards further microbial challenges by an underlying infection.While mice that had received no mAb or conT2 30 min prior to injectionsuccumbed to lethal shock within 24 h, mice treated with T2.5 survived(FIG. 6a ). Intending to employ a complex challenge mimicking infectionfor a distinct shock model, the inventors took advantage of the findingthat shock induction by viable or heat inactivated Gram-positive B.subtilis bacteria is TLR2 dependent not only in a low dose, but also ina high dose model (unpublished observation). Mice were pretreated withT2.5 or conT2 followed by challenge with a lethal dose of B. subtilis(protective protocol). In a separate group of mice, the inventors firstadministered B. subtilis and applied T2.5 up to 3 h later (therapeuticprotocol). In the absence of T2.5 the high dose B. subtilis challengewas lethal for all mice tested (FIG. 6b ). However, when given T2.5either prior (1 h), or up to 2 h after microbial challenge, all B.subtilis challenged mice survived. Most notably, treatment with T2.5even 3 h after potentially lethal injection saved 75% of mice challenged(FIG. 6b ).

The results indicated herein show a therapeutically useful function ofan antagonistic TLR2 mAb in TLR2 driven septic shock. The inventorsfound that application of TLR2 agonists was lethal in two experimentalmodels of septic shock and therefore aimed at identification ofantibodies blocking TLR2. Here the inventors show that the mAb T2.5prevents P₃CSK₄, a synthetic analogue of bacterial lipopeptides, orGram-positive bacteria (B. subtilis) induced shock in mice. T2.5 alsoblocks human TLR2 function, since subcellular NF-κB translocation uponTLR2 specific challenge of primary human macrophages was inhibited uponits application.

The lack of TLR functions negatively affects humans at least upon acuteinfections^(28,29). In a systemic model of polymicrobial sepsisencompassing standardized influx of the gut flora into the peritonealcavity, however, mice benefit from the lack of TLR functions³⁰indicating TLR dependent mediation of harmful effects in acuteinfection. Indeed, blockade or application of LPS binding proteins suchas CD 14, bactericidal/permeability-increasing protein (BPI), or LPSbinding protein (LBP) has been effective to inhibit LPS inducedpathology³¹⁻³⁴ even after LPS application. Since this exemplified theprevalent role of a single cellular system for specific recognition of amicrobial product^(35,36), the inventors attempted to intervene in cellactivation by TLR2 specific microbial products. The finding that T2.5recognized murine and human TLR2 on the cell surface of and within cellscultured in vitro, as well as exhibited antagonistic effects uponapplication to cells expressing these receptors provided a basis for ourattempt. Furthermore, the antagonistic function was specific and dosedependent.

The inventors analyzed the potential of T2.5 to prevent TLR2 drivenimmunopathology. Application of T2.5 30 min or 1 h prior to applicationof lethal doses of TLR2 specific agonists P₃CSK₄ to sensitized mice orof B. subtilis to normal mice, respectively, protected mice againsttheir lethal effects (FIG. 6a and b ), but not against the lethaleffects of LPS (data not shown). In fact, B. subtilis induced shock wasprevented upon application of T2.5 2 h, or even 3 h after shockinduction (100% or 75% of survival, respectively). Their resultsindicate complement mediated depletion of TLR2⁺ cells as an unlikelymechanism of T2.5 mediated shock prevention, since application of themTLR2 specific isotype matched mAb conT2 in vivo did not result inprotection. This implicates reversibility of mAb mediated TLR2 blockadewhich is potentially important for timely recovery of TLR2 dependentcellular responsiveness in later phases of sepsis at which diminishedimmune function is fatal⁹. The demonstration of beneficial effects ofT2.5 in both, a sensitization dependent and a high dose TLR2 specificexperimental model, validate a therapeutical application in the face ofmany animal models being restricted to one of the two respectivedosages. This characteristic may improve transferability of the resultsfrom our animal study to treatment of sepsis in humans⁹.

Perhaps it is the surprisingly very low constitutive surface expressionof TLR2 in host cells such as CD11b⁺ (macrophage), GR1⁺ (granulocyte),CD19⁺ (B cell), CD11c⁺ (dendritic cell) splenocytes, and peritonealcells in vivo (FIG. 4 and data not shown), which explains the efficacyof T2.5 mediated prevention of shock triggered via TLR2. This lowsurface expression is in contrast with relatively high surfaceexpression in unchallenged primary murine (FIG. 1), as well as humanmyeloid cells upon in vitro culture³⁷ giving account for immediate TLR2expression analysis ex vivo. However, comparison of TLR2-staining ofnon-permeabilized and permeabilized cells indicates that a major portionof TLR2 was localized in the intracellular compartment of murine CD11b⁺and GR1⁺ cells, as well as human macrophages (FIG. 4c , unpublishedobservation and FIG. 2b ). In fact, the inventors noted increasedsurface and intracellular TLR2 expression in specific cell populations24 h after bacterial infection which was similar upon LPS challenge(FIG. 4 and data not shown). The time course of TLR2 regulation indistinct immune cells upon microbial contact needs to be investigated inmore detail because it might determine the time frame in which TLRblockade based intervention strategies can be effective.

Antagonistic properties have recently been demonstrated in vitro alsofor two anti human TLR2 mAbs^(38,39) possibly indicating distinct activecomplex formation of TLRs as compared to receptors for which agonisticantibodies have been identified. However, T2.5 interferes with thelipopeptide/TLR2 complex that induces cell activation, as well asrecognizes a human TLR2 construct lacking the N-terminal third of theTLR2LRR rich domain (FIG. 17c ) the inventors found to be dispensablefor cellular recognition of lipopeptides⁴⁰. Thus, the epitope recognizedby T2.5 must be located within the C-terminal portion of the TLR2ECD.The inventors expect that identification of the epitope will show itsconservation between mice and humans. The potential of T2.5, forinstance in combination with further inhibitors of inflammatoryprocesses, to inhibit pathogenesis of clinically important infectionsawaits its evaluation. In conclusion, the data provided herein are thefirst to point out the potential of TLR2 specific antibody applicationas a therapeutic strategy to block TLR2 mediated cell activation in thecourse of acute infection through in vivo evidence.

Methods

Material. Over night (o. n.) B. subtilis (DSMZ.1087) cultures containingapproximately 1×10¹⁰ colony forming units (cfu)/ml (brain heart medium)were used immediately or heat inactivated (h. i.) at 56° C. for 50 min.Synthetic P₃CSK₄ was purchased from ECHAZ microcollections (Tuebingen,Germany), ultra pure LPS from Salmonella minnesota Re595 was from ListLaboratory (Campbell, Calif.), recombinant murine IFNγ and IL-1β fromPeprotech (London, England), and D-galactosamine from Sigma(Deisenhofen, Germany).

Mice. Matched groups of wild-type (TLR2^(+/+)) B57BL/6 and TLR2^(−/−27)mice kindly provided by Tularik (generated by Deltagen; South SanFrancisco, Calif.; nine-fold crossed towards B57BL/6 background) wereapplied.

Generation of TLR2ECD specific antibodies and ELISA. A cDNA fragmentencoding the N-terminal 587 amino acids of mTLR2⁴¹ was amplified from aRAW264.7 cDNA library (advantage kit, BD Clontech, Heidelberg, Germany).The murine TLR2ECD was fused to a C-terminal thrombin cleavage sitefollowed by a human IgGFcγ moiety. The murine TLR2ECD protein waspurified upon overexpression in HEK293 cells and thrombin digestion. ATLR2^(−/−) mouse was immunized by intraperitoneal (i. p.) injection of50 μg of TLR2ECD and 10 nmol of a thioated DNA oligonucleotide(5′-TCCATGACGTTCCTGA-3′, Tib Molbiol, Berlin, Germany) (SEQ ID NO: 5)for three times within eight weeks. Its splenocytes were fused withmurine P3X cells and hybridomas were selected⁴². MAb specificities forTLR2ECD, as well as cyto- and chemokine concentrations in cellsupernatants or murine sera (see below) were analyzed by ELISA (R&Dsystems, Minneapolis, Minn.).

Flow cytometry. Stably transfected HEK293 cell clones, as well asuninduced peritoneal wash-out macrophages were cultured o. n. asdescribed⁴⁰. Flow cytometry was performed upon staining with eitherT2.5, or affinity purified polyclonal rabbit antisera specific for themurine TLR2ECD⁴³ or the Flag tag (Sigma), as well as respectivesecondary mAbs (BD Pharmingen, Heidelberg, Germany).

For establishment of mTLR2 expression analysis in primary cells, surfaceand intracellular T2.5 dependent staining of CD11b⁺ splenocytes⁴² fromwild-type and TLR2^(−/−) mice challenged with LPS (0.5 mg, i. p., 24 h)were compared by flow cytometry (CyAn, Dako Cytomation, Fort Collins,Colo.). Cells were stained with photoactivated ethidium monoazide(Molecular Probes, Amsterdam, Netherlands) immediately upon isolation,followed by TLR2 specific surface staining, or intracellular staining(cytofix/cytoperm, BD Pharmingen). In order to analyze TLR2 expressionin non- or B. subtilis infected (5×10¹⁰ cfu, i. p., 24 h) mice,peritoneal washout cells and splenocytes⁴² from five wild-type orTLR2^(−/−) mice were pooled, respectively. Fluorescence labeled cellsurface marker antibodies (BD Pharmingen) and primary T2.5 stained withsecondary anti mIgG1 were used as indicated.

Immunoprecipitation and immunoblot analysis. Lysates of Flag-TLR2transfected HEK293 cells or macrophages, as well as 1 μg of antibody andprotein G beads (Santa Cruz, Calif.) were mixed for o. n. precipitation.Immune complexes or cell lysates were analyzed by immunoblot analysis asdescribed⁴⁰. Precipitations were controlled by application of Flagspecific (mAb M2, Sigma) or protein G beads only. Flag (HEK293) ormTLR2⁴³ (RAW264.7) specific antisera were applied for immunoblotanalyses. In contrast, total lysates of macrophages (see inhibitionexperiments) were analyzed for phosphorylation of kinases indicated.

Cytochemical staining of TLR2 or NF-κB. Transfected HEK293 cell clones,as well as primary murine or human macrophages, the last isolated as CD14⁺ peripheral blood leukocytes and cultured in 20% of autologousserum⁴⁴, were grown on slides. Cells were washed with PBS,permeabilized, and incubated with 50 μg/ml TLR2 specific mAb and/or antiNF-κB/p65 (polyclonal rabbit, Santa Cruz)⁴⁰. Specific secondary αIgGantibodies were applied. Cell membranes were stained with labeledconcanavalin A (Molecular Probes).

Inhibition of TLR2 dependent cell activation in vitro and in vivo.Transiently transfected HEK293 cells, murine RAW264.7, as well asprimary macrophages were used⁴⁰. 50 μg/ml of antibodies were applied 30min prior to challenge with 100 ng/ml of LPS, IL-1β, P₃CSK₄, or 1×10⁶cfu/ml of h. i. B. subtilis. HEK293 cells were cotransfected withreporter⁴⁵, human CD14, human or mTLR2, and MD2 (provided by Tularik,Drs. Golenbock and Heine, as well as Miyake, respectively) expressionplasmids, and NF-κB dependent reportergene activity was assayed after 6h of stimulation⁴⁰. TNFα concentrations in supernatants of RAW264.7 andprimary murine macrophages, as well as NF-κB translocation in humanmacrophages⁴⁴ were analyzed 24 h and 90 min after challenge,respectively. For carrying out challenge and antibody dose dependentNF-κB dependent EMSA, as well as p38, Erk1/2, and Akt phosphorylationspecific immunoblot analysis (Cell signaling, Frankfurt, Germany),RAW264.7 macrophages were used. 1×10⁶ cells were pretreated withantibodies as described above at various concentrations and stimulatedfor 90 min (EMSA) or 30 min (kinase phosphorylation analysis)⁴⁰. Foranalysis of TLR2 inhibition in vivo, mice were injected i. p. with 1 mgof T2.5 or left untreated. 1 h later, 100 μg of P₃CSK₄ and 20 mg ofD-galactosamine were injected i. p. Serum concentrations of TNFα, IL-8,IL-6, and IL-12p40 in five unchallenged control mice were 0.05 ng/ml,0.43 ng/ml, not detectable, and 0.44 ng/ml, respectively.

Systemic shock induction. In an experimental sensitization dependentmodel²⁷, mice were injected intravenously with 1.25 μg of murine IFNγ.20 min later, mice were injected i. p. with 1 mg of mAb as indicated. 50min after IFNγ injection, 100 μg of synthetic P₃CSK₄ and 20 mg ofD-galactosamine were injected i. p. as well. The experimental high doseshock model encompassed a single i. p. injection of h. i. B. subtilissuspension (corresponding to 5×10¹⁰ cfu) with prior (1 h) or subsequent(1 h, 2 h, or 3 h) i. p. injection of 1 mg mAb as indicated.

Material and Method (for FIG. 17)

Abrogation of TLR2ECD ligand-binding by T2.5 and analysis of T2.5epitope localization. To investigate whether T2.5 blocked binding ofTLR2 to its synthetic agonist P₃CSK₄ the inventors established a SPRbiosensor based binding assay. P₃CSK₄ was immobilized on a chip surfaceand binding of T2EC was tested under various conditions. PHCSK₄, anon-active analogue of P₃CSK₄, was used as control and sensograms aredisplayed as subtracted binding curves. Binding of T2EC to P₃CSK₄ wasspecific (FIG. 17a ). When T2EC was pre-incubated with T2.5, theantibody dose dependently inhibited T2EC-P₃CSK₄ binding (FIG. 17a ). Amolar ratio of 3.3 (T2.5:T2ECD) was required to reduce binding to 50%.Pre-incubation of T2EC with T2.5 at a 10 fold molar excess abrogatedT2EC-P.₃CSK₄ interaction (FIG. 17a ). In contrast, an isotype matchedcontrol antibody did not block binding of T2EC to P₃CSK₄ even if appliedat 10 fold molar excess (FIG. 17b ). If applied alone, both mAbs did notinteract with the sensor-chip surface (FIG. 17, a and b). The N-terminalthird of the LRR-rich domain of human TLR2 is not involved inlipopeptide recognition (37) and T2.5 cross-reacts with human TLR2(FIGS. 2b and 3e ). Thus, we applied T2.5 to HEK293 cells overexpressinga mutant construct of human TLR2 lacking the respective portion of thewild-type ECD (37). Specific abrogation of NF-κB dependent reporter geneactivation upon P₃CSK₄ challenge after administration of T2.5 stronglysuggests localization of the epitope recognized by T2.5 within theC-terminal portion of the TLR2ECD (FIG. 17c ).

Direct interaction between TLR2 and P₃CSK₄ was demonstrated and allowedcomparison of TLR2 and TLR2-T2.5-complex affinities to this ligand. SPRanalysis showed the direct and specific interaction between TLR2ECD andP3CSK₄, as well as a specific and dose dependent inhibition of thisinteraction by T2.5 (FIG. 17, a and b), indicating that binding of T2.5masked the ligand-binding domain in TLR2. Accordingly, T2.5 antagonizednot specifically P₃CSK₄, but also h. i. B. subtilis, PGN, LTA, andmycoplasmal macrophage activating protein induced TLR2-dependent cellactivation (FIG. 3 and data not shown). Blockage was specific and dosedependent (FIG. 3). Taken together, these findings show that specificbinding of ligands to a discrete site within the TLR2ECD is aprerequisite for TLR2 mediated signaling.

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The invention claimed is:
 1. An isolated cross-reactive antibody or afragment thereof, which specifically inhibits or blocks mammalianToll-like Receptor 2 (TLR2)-mediated immune cell activation byspecifically binding to the C-terminal portion of the extracellulardomains of at least human and murine TLR2, wherein the antibody orfragment thereof specifically binds through the variable regions of theheavy and light chains, wherein the heavy chain variable regioncomprises a complementarity determining region 1 (CDR1) region whereinthe amino acid sequence of the CDR1 region comprisesGly-Phe-Thr-Phe-Thr-Thr-Tyr-Gly (residues 58-65 of SEQ ID NO:6), a CDR2region wherein the amino acid sequence of the CDR2 region comprisesIle-Tyr-Pro-Arg-Asp-Gly-Ser-Thr (residues 83-90 of SEQ ID NO: 6) and aCDR3 region wherein the amino acid sequence of the CDR3 region comprisesAla-Arg-Leu-Thr-Gly-Gly-Thr-Phe-Leu-Asp-Tyr (residues 129-139 of SEQ IDNO:6), and wherein the light chain variable region comprises a CDR1region wherein the amino acid sequence of the CDR1 region comprisesGlu-Ser-Val-Glu-Tyr-Tyr-Gly-Thr-Ser-Leu (residues 47-56 of SEQ ID NO:7),a CDR2 region wherein the amino acid sequence of the CDR2 regioncomprises Gly-Ala-Ser (residues 74-76 of SEQ ID NO:7) and a CDR3 regionwherein the amino acid sequence of the CDR3 region comprisesGln-Gln-Ser-Arg-Lys-Leu-Pro-Trp-Thr (residues 113-121 of SEQ ID NO:7),wherein the CDRs are identified by the IMGT database.
 2. The antibody orantibody fragment of claim 1, wherein the antibody is selected from thegroup consisting of a polyclonal antibody, a monoclonal antibody, ahumanised antibody, a chimeric antibody and a synthetic antibody.
 3. Theantibody or fragment thereof of claim 1 wherein the antibody comprises:a heavy chain variable region having the amino acid sequence of SEQ IDNO:6; a light chain variable region having the amino acid sequence ofSEQ ID NO:7; or both.
 4. The antibody fragment of claim 1 comprisingcomplementarity determining regions (CDRs) of the heavy chain variabledomain, wherein the amino acid sequence of the CDR1 region comprisesGly-Phe-Thr-Phe-Thr-Thr-Tyr-Gly (residues 58-65 of SEQ ID NO:6), theamino acid sequence of the CDR2 region comprisesIle-Tyr-Pro-Arg-Asp-Gly-Ser-Thr (residues 83-90 of SEQ ID NO:6) and theamino acid sequence of the CDR3 region comprisesAla-Arg-Leu-Thr-Gly-Gly-Thr-Phe-Leu-Asp-Tyr (residues 129-139 of SEQ IDNO:6), and the complementarity determining regions (CDRs) of the lightchain variable domain, wherein the amino acid sequence of the CDR1region comprises Glu-Ser-Val-Glu-Tyr-Tyr-Gly-Thr-Ser-Leu (residues 47-56of SEQ ID NO:7), the amino acid sequence of the CDR2 region comprisesGly-Ala-Ser (residues 74-76 of SEQ ID NO:7) and the amino acid sequenceof the CDR3 region comprises Gln-Gln-Ser-Arg-Lys-Leu-Pro-Trp-Thr(residues 113-121 of SEQ ID NO:7), wherein the CDRs are identified bythe IMGT database.
 5. The antibody fragment of claim 1 wherein theantibody fragment is selected from the group consisting of a Fabantibody fragment, a F(ab′)2 antibody fragment and an Fv antibodyfragment.
 6. An isolated nucleic acid, which comprises nucleic acidsNos. 172-195, 247-270 and 385-417 of SEQ ID NO:1; nucleic acids Nos.139-168, 220-228 and 337-363 of SEQ ID NO:2; or both nucleic acids Nos.172-195, 247-270 and 385-417 of SEQ ID NO:1 and nucleic acids Nos.139-168, 220-228 and 337-363 of SEQ ID NO:2.
 7. The isolated nucleicacid of claim 6, said isolated nucleic acid further comprising a nucleicacid encoding one or more regulatory sequences operably linked thereto.8. An isolated cross-reactive antibody or a fragment thereof, whichspecifically inhibits or blocks mammalian Toll-like Receptor 2(TLR2)-mediated immune cell activation by specifically binding to theC-terminal portion of the extracellular domains of at least human andmurine TLR2, wherein the antibody or fragment thereof specifically bindsthrough the variable regions of the heavy and light chains, wherein theheavy chain variable region comprise a complementarity determiningregion 1 (CDR1) region wherein the amino acid sequence of the CDR1region comprises Thr-Tyr-Gly-Ile-Asn (residues 63-67 of SEQ ID NO:6), aCDR2 region wherein the amino acid sequence of the CDR2 region comprisesTrp-Ile-Tyr-Pro-Arg-Asp-Gly-Ser-Thr-Asn-Phe-Asn-Glu-Asn-Phe-Lys-Asp(residues 82-98 of SEQ ID NO: 6), and a CDR3 region wherein the aminoacid sequence of the CDR3 region comprisesAla-Arg-Leu-Thr-Gly-Gly-Thr-Phe-Leu-Asp-Tyr (residues 129-139 of SEQ IDNO:6), and wherein the light chain variable region comprises a CDR1region wherein the amino acid sequence of the CDR1 region comprisesArg-Ala-Ser-Glu-Ser-Val-Glu-Tyr-Tyr-Gly-Thr-Ser-Leu-Met-Gln (residues44-58 of SEQ ID NO:7), a CRD2 region wherein the amino acid sequence ofthe CDR2 region comprises Gly-Ala-Ser-Asn-Val-Glu-Ser (residues 74-80 ofSEQ ID NO:7) and a CDR3 region wherein the amino acid sequence of theCDR3 region comprises Gln-Gln-Ser-Arg-Lys-Leu-Pro-Trp-Thr (residues113-121 of SEQ ID NO:7).
 9. The antibody or antibody fragment of claim8, wherein the antibody is selected from the group consisting of apolyclonal antibody, a monoclonal antibody, a humanised antibody, achimeric antibody and a synthetic antibody.
 10. The antibody or fragmentthereof of claim 8, wherein the antibody comprises: a heavy chainvariable region having the amino acid sequence of SEQ ID NO:6; a lightchain variable region having the amino acid sequence of SEQ ID NO:7; orboth.
 11. The antibody fragment of claim 8 comprising complementaritydetermining regions (CDRs) of the heavy chain variable domain, whereinthe amino acid sequence of the CDR1 region comprises Thr-Tyr-Gly-Ile-Asn(residues 63-67 of SEQ ID NO:6), the amino acid sequence of the CDR2region comprisesTrp-Ile-Tyr-Pro-Arg-Asp-Gly-Ser-Thr-Asn-Phe-Asn-Glu-Asn-Phe-Lys-Asp(residues 82-98 of SEQ ID NO: 6), and the amino acid sequence of theCDR3 region comprises Ala-Arg-Leu-Thr-Gly-Gly-Thr-Phe-Leu-Asp-Tyr(residues 129-139 of SEQ ID NO:6), and the complementarity determiningregions (CDRs) of the light chain variable domain, wherein the aminoacid sequence of the CDR1 region comprisesArg-Ala-Ser-Glu-Ser-Val-Glu-Tyr-Tyr-Gly-Thr-Ser-Leu-Met-Gln (residues44-58 of SEQ ID NO:7), the amino acid sequence of the CDR2 regioncomprises Gly-Ala-Ser-Asn-Val-Glu-Ser (residues 74-80 of SEQ ID NO:7)and the amino acid sequence of the CDR3 region comprisesGln-Gln-Ser-Arg-Lys-Leu-Pro-Trp-Thr (residues 113-121 of SEQ ID NO:7).12. The antibody fragment of claim 8 wherein the antibody fragment isselected from the group consisting of a Fab antibody fragment, a F(ab′)2antibody fragment and an Fv antibody fragment.
 13. An isolated nucleicacid, which comprises nucleic acids Nos. 187-201, 244-294 and 385-417 ofSEQ ID NO:1; nucleic acids Nos. 130-174, 220-240 and 337-363 of SEQ IDNO:2; or both nucleic acids Nos. 187-201, 244-294 and 385-417 of SEQ IDNO:1 and nucleic acids Nos. 130-174, 220-240 and 337-363 of SEQ ID NO:2.14. The isolated nucleic acid of claim 13, said isolated nucleic acidfurther comprising a nucleic acid encoding one or more regulatorysequences operably linked thereto.
 15. An isolated cross-reactiveantibody or a fragment thereof, which specifically inhibits or blocksmammalian Toll-like Receptor 2 (TLR2)-mediated immune cell activation byspecifically binding to the C-terminal portion of the extracellulardomains of at least human and murine TLR2, wherein the antibody orfragment thereof specifically binds through the variable regions of theheavy and light chains, wherein the heavy chain variable regioncomprises amino acid residues Nos. 58-65(Gly-Phe-Thr-Phe-Thr-Thr-Tyr-Gly), amino acid residues Nos. 83-90(Ile-Tyr-Pro-Arg-Asp-Gly-Ser-Thr) and amino acid residues Nos. 129-139(Ala-Arg-Leu-Thr-Gly-Gly-Thr-Phe-Leu-Asp-Tyr) of SEQ ID NO:6, andwherein the light chain variable region comprises amino acid residuesNos. 47-56 (Glu-Ser-Val-Glu-Tyr-Tyr-Gly-Thr-Ser-Leu), amino acidresidues Nos. 74-76 (Gly-Ala-Ser) and amino acid residues Nos. 113-121(Gln-Gln-Ser-Arg-Lys-Leu-Pro-Trp-Thr) of SEQ ID NO:7.
 16. The antibodyor antibody fragment of claim 15, wherein the antibody is selected fromthe group consisting of a polyclonal antibody, a monoclonal antibody, ahumanised antibody, a chimeric antibody and a synthetic antibody. 17.The antibody fragment of claim 15 comprising amino acid residues Nos.58-65 (Gly-Phe-Thr-Phe-Thr-Thr-Tyr-Gly), amino acid residues Nos. 83-90(Ile-Tyr-Pro-Arg-Asp-Gly-Ser-Thr) and amino acid residues Nos. 129-139(Ala-Arg-Leu-Thr-Gly-Gly-Thr-Phe-Leu-Asp-Tyr) of SEQ ID NO:6, and aminoacid residues Nos. 47-56 (Glu-Ser-Val-Glu-Tyr-Tyr-Gly-Thr-Ser-Leu),amino acid residues Nos. 74-76 (Gly-Ala-Ser) and amino acid residuesNos. 113-121 (Gln-Gln-Ser-Arg-Lys-Leu-Pro-Trp-Thr) of SEQ ID NO:7. 18.The antibody fragment of claim 15 wherein the antibody fragment isselected from the group consisting of a Fab antibody fragment, a F(ab′)2antibody fragment and an Fv antibody fragment.